In this project, we have continued our efforts to establish how the skeleton and in particular the skeletal stem/progenitor population is maintained and differentiates within the confines of an actively remodeling extracellular matrix. To this end, we have extended our work to understand how the various progeny of the skeletal stem cells and osteoclastic cells, identified by distinct differentiation stage-specific gene expression rely on Mt-Mmp-dependent proteolysis for both physical matrix remodeling as well as modulation of cell-signaling molecules to maintain skeletal homeostasis. Using mice in which Mt1-Mmp was deleted in cells at different stages of osteogenic maturation, it was determined that Mt1-Mmp expression is essential for removal of Rank1 from the surface of early (OSX-Cre) osteoprogenitor cells (Purcell and Kaplan, co-first authors, et al, submitted to PLoS One). We have further characterized the phenotype of mice in which Mt1-Mmp was deleted from fully mature Osteocalcin-expressing cells (Ocn-Cre mice). These mice are born overtly normal, but develop an osteopenic phenotype that is not due to increased osteoclastogenesis as seen in the Osx-Cre mice. The mice begin to spontaneously fracture at 6 wks, and instead of a normal fracture callus and red marrow, the callus and marrow cavity are completely filled with yellow, adipocyte-rich marrow. Expression analyses of marrow-ablated bone from these mice and control littermates demonstrated a dramatic upregulation of the transmembrane protein delta-like homolog, Dlk1. In vitro, BMSCs derived from the global Mt1-Mmp-deficient mice displayed increased adipogenic differentiation, similar to what is made by Dlk1-deficient BMSCs cells obtained from our collaborators, Drs Steven Bauer and Heba Degheidy from the FDA. In the absence of Mt1-Mmp, Dlk1 was highly enriched in osteogenic cells, the majority of which was membrane-bound. We demonstrated by co-expression that Dlk1 is processed into a soluble form by Mt1-Mmp, and further, that this form of Dlk1 efficiently inhibited the induction of Notch signaling by Delta-like 4 (Dll4), one of the canonical notch ligands. Our results demonstrate that adipogenic expansion in the bone marrow is coincident with a loss of efficient cleavage of the membrane form of Dlk1 into a soluble adipogenic suppressor by Mt1-Mmp. Taken together, these observations highlight the essential role of Mt1-Mmp-mediated proteolysis of Dlk1 as a gatekeeper of default differentiation of SSCs/BMSCs into marrow adipocytes. Equally important, these findings underscore the essential role undertaken by mature bone cells (Ocn+) in the regulation of SSC/BMSC fate and maintenance of multipotency through Dlk1-mediated suppression of adipogenic differentiation. This project is now being performed in colllaboration with our former K99/R00 fellow, Dr. Jason A. Horton, and our FDA colleagues.